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Abstract

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe a protocol to prepare whole cell extracts (WCE) and short term culture filtrate (CF) from Mtb or the vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) (Mehra et al., 2013). These are both slow growing mycobacteria, but the same basic procedure can easily be adapted to analyze secreted proteins from rapidly growing mycobacteria, such as Mycobacterium smegmatis (Msmeg), a non-pathogenic species commonly used in the laboratory. The fractions obtained can be analyzed by western blotting to examine proteins of interest or by mass spectrometry if antibodies are not available or to examine the entire secretome. Genetic knockout mutants for the gene of interest serve as a negative control. Additionally, levels of a cytosolic protein such as the chaperone GroEL or the pyruvate dehydrogenase E2 component sucB (Rv2215/dlaT) should be assessed in the CF fraction to rule out the possibility that a positive signal in CF is due to bacterial lysis (see Figure 1). By varying the growth conditions of the strain, this in vitro secretion assay can be used to examine conditions that alter the secretome. We are thankful to Magnus Stiegedal for helpful tips on TCA (trichloroacetic acid) precipitation.

Figure 1. Western analysis of secretion of EsxH by BCG. BCG containing an empty vector control and EsxG-EsxH-FLAG expression construct (FLAG tag at C terminal of EsxH) were analyzed for presence EsxH by anti-FLAG western in WCE and CF prepared as described in the protocol. DlaT was used as a loading control to indicate the degree of bacterial lysis.

Materials and Reagents

Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility.

15 ml falcon tubes (Corning, catalog number: 430052 ) with plug seal capsNote: These 50 ml tubes are compatible with organic solvents and high speed centrifugation. Falcon tubes with these features can be used from different vendors.

Inoculate liquid cultures of mycobacteria from frozen stocks, prepared
from mid-log phase mycobacterial cultures (OD600 between 0.5 to 1.0)
that were frozen in 18% glycerol at -80 °C.

For inoculation,
the doubling rate of the mycobacterial species needs to be kept in mind.
Msmeg doubles approximately every 3 h, whereas BCG and Mtb double
approximately every 20 h in 7H9 complete media at 37 °C.

The
cultures are inoculated in 10 ml of 7H9 complete media in 30 ml square
media bottles. Include appropriate antibiotics if required e.g. antibiotics can be included at appropriate concentration for selecting a
plasmid when growing a transformed mycobacterial strain.

The
mycobacterial cultures are incubated at 37 °C with slow shaking at a
speed of 90 rpm. BCG and Mtb cultures are placed in aerosol containment
in a shaking incubator.

For Msmeg, inoculate 200 µl from
glycerol stock and grow overnight to saturation. Dilute the over-night
culture 1:40 into fresh 7H9 complete media and grow to mid-log phase.
For BCG and Mtb, inoculate 0.5 ml from frozen stock. It will take
several days (~4 days) for the culture to reach mid-log phase and the
exact time depends upon the initial inoculum density and growth
conditions.

Measure the OD600 of the culture. Determine the
culture volume required to give a final starting OD600 of at least 0.25
when resuspended in 25 ml of Sauton’s media [culture volume in ml = 25
ml x desired OD600 (e.g. 0.25)/OD600 of the experimental culture].

Transfer this volume of culture media to a 15 ml falcon tube and
pellet the bacterial cells by centrifuging at 1500 x g for 7 min at room
temperature. Remove the supernatant and resuspend the pellet in PBS.
Again centrifuge at 1,500 x g for 7 min. This wash step is important to
remove albumin carried over from 7H9 complete media. Notes:

Sauton’s media is a minimal media in which mycobacteria grow more slowly than they do in 7H9 complete media.

Alternatively, if the effect of metal ions on secretion is to be
studied, one can use chelated Sauton’s media as the minimal media and
add defined concentrations of metal ions.

Monitor OD600 of the
culture. Grow the culture to between 0.5 to 0.8 OD600. It takes about 6 h
for Msmeg and 48 h for BCG and Mtb This procedure could be modified to
inoculate the bacteria at a lower density and allow them to grow for a
longer period of time, depending upon the goals of the experiment.

Harvesting the bacterial culture and preparation of the WCE

After incubating in Sauton’s media as described above, determine the OD600.

Calculate the volume corresponding to 5 OD600 units. For example, if
the OD600 of the culture is 0.5, then 10 ml will correspond to 5 OD
units (desired volume = 5/OD600 of the experimental culture).

Transfer 5 OD600 of the culture into a 15 ml falcon tube (to be used to
prepare the WCE). Transfer the remaining culture into a 50 ml falcon
tube (to be used to precipitate proteins from the CF). Centrifuge both
tubes at 2,000 x g for 5 min at 4 °C.

Pool the supernatant
from both the 15 and 50 ml falcons into one unused 50 ml falcon tube and
store on ice until you are ready to process it further (see below,
preparation of CF in step C).

Wash the pellet in the 15 ml falcon with 5 ml of PBS by resuspending and centrifuging at 2,000 x g, 5 min, 4 °C.

Discard the supernatant and resuspend the pellet again in 1 ml of PBS
and transfer it to a 2 ml Sarstedt tube with O ring in the screw cap
(for containing aerosols during bead beating).

Spin on a
table top centrifuge at 3,000 x g, 5min and discard the supernatant. To
the pellet add 300 µl of protein extraction buffer with protease
inhibitors. Resuspend the pellet by pippetting.

Dispense 100
µl of 0.1 mm zirconia/silica beads into a 1.7 ml eppendorf and add this
to the Sarstedt tube containing the pellet.

Using a bead
beater contained within a class II bio-safety cabinet (please read the
bead beater manual for the operating procedure and also see the link
provided in Equipment, item 19), bead beat the sample for 1 min at 3,450
oscillations/min and place it on ice for 2 min to cool. Repeat this
twice. This sample is the WCE. Centrifuge the lysate at full speed for 2
min in a microcentrifuge to settle the froth before opening the tube. Notes:

Alternate bead beaters may have different speeds and hence the user
should optimize bead beating (speed and time) for maximal lysis of
mycobacterial pellet.

The degree of lysis can be established by plating the bacteria before and after bead beating for colony forming units.

For SDS-PAGE, add 60 µl of 5x SDS-PAGE sample buffer to the Sarstedt
tube and then gently mix by inversion. Incubate at 95 °C for 5 min. The
heating step helps to denature the sample and also sterilize
mycobacterial lysate after bead beating. The mycobacterial WCE can be
stored at -80 °C. Note: The WCE of Mtb can generally be transferred
out of BSL3 facility at this step as long as that is consistent with the
institutional standards of practice.

Before use, thaw at room
temperature and then heat at 95 °C for 5 min. 20 µl of the WCE is often
sufficient for SDS-PAGE and western analysis, although this amount will
depend upon the target protein levels and the sensitivity of the
detection method.

Alternatively, it can be filtered by vacuum using 50 ml Steriflip-GV
filter units (0.22 µM) to remove any bacteria from the supernatants.

This can be done twice depending on the turbidity of the supernatant.

Double filtration may also be required depending upon the standard
operating procedures of the facility to transfer the CF from the BSL3
facility.

Pre-chill trichloroacetic acid (TCA) on ice. Add
TCA to a final concentration of 12% to the CF (3 ml of 100% TCA to the
25 ml sample). Invert the tube several times to mix and put the falcon
tube at 4 °C overnight. Ensure the falcon tubes used for precipitation
are compatible with organic solvents and high speed centrifugation.

Spin the CF at 15,000 x g for 15 min at 4 °C.

Discard the supernatant carefully to avoid loosing the pellet.

Wash the remaining TCA by adding 5 ml acetone (pre-chilled at 4 °C) slowly along the sides of the tube.

Spin at 15,000 x g for 15 min at 4 °C.

Discard the supernatant very carefully at this stage since the pellet is not adhered tightly to the tube.

Leave the tubes open in a Class II BSC for 10-20 min for faster drying
of the pellet. Do not over dry to avoid difficulty in solubilizing of
the pellet.

Resuspend the pellet in 150 µl 1x SDS-PAGE
sample buffer. You may need to place tubes in a 65 °C water bath briefly
to completely resuspend.

If samples turns yellow due to
incomplete removal of TCA, add a few microliters of 1 M Tris-HCl (pH 8)
until they turn blue again.

Store sample at -70 °C. Thaw
before use. 10 µl of the sample may be enough for SDS-PAGE and western
analysis, although the amount will depend upon the amount of the target
protein and the sensitivity of the detection method.

Notes

For applications incompatible with TCA precipitations, Centricon Plus-70 centrifugal filter units can be used to concentrate the CF from step B9 as per the manufacturer’s instructions. This can be followed by buffer exchange to desired buffers. Care must be taken to use Centricons with membranes of molecular weight cut-off less than the molecular weight of the target proteins to be analyzed in the CF.

Recipes

7H9 complete media (1 L)
7H9 powder 4.7 g
50% glycerol 4.0 ml
20% Tween 80 2.5 ml
Water to 900 ml
Dissolve 7H9 powder in water and add glycerol and Tween 80
Adjust the amount of water to give a final volume of 900 ml
Add 100 ml of OADC for Mtb or 100 ml of ADC for Msmeg and BCG
Sterilize through 0.22 µM filter and stored at 4 °C (the media can be stored for up to six months. If supplemented with additional additives, like antibiotics or salts, the media should be stored as appropriate for the compounds if they are sensitive to light or degradation)Note: Alternatively, autoclave after dissolving 7H9 and glycerol in 897.5 ml water and then supplement with sterile tween-80 and 100 ml of ADC/OADC and store at 4 °C as noted above.

Chelated Sauton’s media (1 L)
Potassium phosphate, monobasic 0.5 g
L-asparagine monohydrate 4.0 g
Citric acid monohydrate 2.2 g
Glycerol 60 ml
20% Tween 80 2.5 ml
Water to 1 L
Chelex 100 resin 10 g
Dissolve the components in water
Adjust the pH to 7.4 with 5 N NaOH and make up the volume to 1 L with water
Add 10 g Chelex 100 resin and stir the media for 1-2 days at room temperature
Sterilize through a 0.22 µM filter unit
Dissolve 1 g of MgSO4.7H2O in 5 ml water and sterilize it by filtration and add to the sterilized media
Stored the media at 4 °C as mentioned above for 7H9 complete media
Defined concentrations of metals like iron, copper and zinc can be added back after chelation.

This protocol was originally used in the published work Mehra et al. (2013). This published work was supported by grants and fellowships from the NIH (R01 AI087682), the Doris Duke Charitable Foundation, the Infectious Disease Society of America, the Michael Saperstein Medical Scholars Research Fund (New York University School of Medicine), Potts Memorial Foundation and the American Society of Microbiology.

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